Apoptosis-inducing polypeptides

ABSTRACT

An isolated water-soluble VP1 polypeptide of foot-and-mouth disease virus and a nucleic acid encoding the polypeptide. Also disclosed are a pharmaceutical composition containing the polypeptide or nucleic acid and related methods of inducing apoptosis and treating an apoptosis-related disorder.

RELATED APPLICATION

This application is a division of and claims priority to U.S.application Ser. No. 10/863,637, filed Jun. 8, 2004, which is acontinuation-in-part of, and claims priority to, U.S. application Ser.No. 10/449,531, filed May 29, 2003, the contents of which areincorporated herein by reference.

BACKGROUND

Apoptosis, i.e., programmed cell death, is a normal physiologicalprocess of a cell, which is characterized by DNA fragmentation,cytoplasma shrinkage, membrane change, and cell death without damagingneighboring cells. This process is regulated by a combination of variousextracellular and intracellular signals. It allows a multicelluarorganism to replace aged cells, control the cell number and the tissuesize, and protect itself from cells that may lead to lethality. See,e.g., Li et al., Science 302, 1560-1563. Impaired apoptosis results inexcessive levels of unwanted cells, which, in turn, cause disorders suchas cancers, autoimmune diseases, immunodeficiency diseases, reperfusioninjuries, and neurodegenerative diseases. Therefore, apoptosis-inducingcompounds are drug candidates for treating these disorders.

SUMMARY

This invention relates to an isolated water-soluble VP1 polypeptide offoot-and-mouth disease virus that can induce apoptosis. The full-lengthVP1 polypeptide and the nucleic acid encoding it (SEQ ID NOs: 1 and 2,respectively) are listed below:

 M  A  S  M   T  G  G   Q  Q  M   G  R  G  S   T  T  S (SEQ ID NO: 1)  1 ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGCGGAT CCACCACCTC (SEQ ID NO:2)   A  G  E   S  A  D  P   V  T  A   T  V  E   N  Y  G  G  51TGCGGGTGAG TCTGCGGACC CCGTGACTGC CACCGTCGAG AACTACGGTG   E  T  Q   V  Q  R   R  Q  H  T   D  S  A   F  I  L 101 GTGAGACACAAGTCCAGAGG CGCCAGGACA CGGACAGTGC GTTCATATTG D  R  F  V   K  V  K   P  K  E   Q  V  N  V   L  D  L 151 GACAGGTTCGTGAAAGTCAA GCCAAAGGAA CAAGTTAATG TGTTGGACCT  M  Q  I   P  A  H  T   L  V  G   A  L  L   R  T  A  T 201 GATGCAGATCCCTGCCCACA CCTTGGTAGG GGCGCTCCTG CGAACGGCCA   Y  Y  F   S  D  L   E  L  A  V   K  H  E   G  D  L 251 CCTACTACTTCTCTGACCTG GAGCTGGCCG TCAAGCACGA GGGCGATCTC T  W  V  P   N  G  A   P  E  T   A  L  D  N   T  T  N 301 ACCTGGGTCCCAAACGGCGC CCCTGAGACA GCACTGGACA ACACTACCAA  P  T  A   Y  H  K  E   P  L  T   R  L  A   L  P  Y  T 351 CCCAACAGCTTACCACAAGG AACCCCTCAC ACGGCTGGCG CTGCCTTACA   A  P  H   R  V  L   A  T  V  Y   N  G  S   S  K  Y 401 CGGCTCCACACCGTCTCTTA GCGACCGTCT ACAACGGGAG CAGTAAGTAC  G  D  T  S   T  N  N   V  R  G   D  L  Q  V   L  A  Q 451 GGTGACACCA GCACTAACAA CGTGAGAGGTGACCTTCAAG TGTTAGCTCA  K  A  E   R  T  L  P   T  S  F   N  F  G   A  I  K  A 501 GAAGGCAGAAAGAACTCTGC CTACCTCCTT CAACTTCGGT GCCATCAAGG   T  R  V   T  E  L   L  Y  R  M   K  R  A   E  T  Y 551 CAACTCGTGTTACTGAACTA CTCTACAGAA TGAAGAGAGC CGAGACATAC  C  P  R  P   L  L  A   I  Q  P   S  D  A  R   H  K  Q 601 TGTCCCAGGCCCCTTCTCGC CATTCAACCG AGTGACGCTA GACACAAGCA R  I  V   A  P  A  K   Q  L  L   L  E  H   H  H  H  H 651 GAGGATTGTGGCACCCGCAA AACAGCTTCT GCTCGAGCAC CACCACCACC   H 701 ACCAC

In one aspect, the invention features an isolated water-soluble VP1polypeptide of foot-and-mouth disease virus that contains RGD (SEQ IDNO: 6). The polypeptide is 25 to 800 amino acids in length (i.e., anynumber between 25 and 800 amino acids, e.g., 29 and 235 amino acids,inclusive). In one embodiment, it contains RGDL (SEQ ID NO: 5) orNGSSKYGDTSTNNVRGDLQVLAQKAERTL (SEQ ID NO: 4). In a preferred embodiment,it contains the sequence of the full-length VP1 polypeptide listed above(SEQ ID NO: 1), the sequence of a mutant form that has a cysteine 201 toserine mutation (SEQ ID NO: 3), or the sequence of capsid polyprotein P1listed below (VP4-1, SEQ ID NO: 7).

(SEQ ID NO: 7) GAGQSSPTTGSQNQSGNTGSIINNYYMQQYQNSMDTQLGDNAISGGSNEGSTDTTSTHTNNTQNNDWFSKLANTAFSGLFGALLADKKTEETTLLEDRILTTRNGHTTSTTQSSVGVTYGYATAEDFVSGPNTSGLETRVVQAERFFKTHLFDWVTSDPFGRCHLLELPTDHKGVYGSLTDSYAYMRNGWDVEVTAVGNQFNGGCLLVAMVPELCSISKRELYQLTLFPHQFINPRTNMTAHITVPYLGVNRYDQYKVHKPWTLVVMVVAPLTVNNEGAPQIKVYANIAPTNVHVAGELPSKEGIFPVACSDGYGGLVTTDPKTADPVYGKVFNPPRNLLPGRFTNLLDVAEACPTFLHFDGDVPYVTTKTDSDRVLAQFDLSLAAKHMSNTFLAGLAQYYTQYSGTINLHFMFTGPTDAKARYMVAYAPPGMEPPKTPEAAAHCIHAEWDTGLNSKFTFSIPYLSAADYAYTASDVAETTNVQGWVCLFQITHGKADGDALVVLASAGKDFDLRLPVDARTQTTSAGESADPVTATVENYGGETQVQRRQHTDIAFILDRFVKVKPKEQVNVLDLMQIPAHTLVGALLRTATYYFSDLELAVKHEGDLTWVPNGAPETALDNTTNPTAYHKEPLTRLALPYTAPHRVLATVYNGSSKYGDTSTNNVRGDLQVLAQKAERTLPTSFNFGAIKATRVTELLYRMKRAETYCPRPLLAIQPSDARHKQRIVAPAKQLL

In one example, the polypeptide of this invention, upon binding to areceptor on a cell, e.g., such as integrin, induces death of the cell.Exemplary cells include an MCF-7 cell, a T-47D cell, a PC-3 cell, a22Rv1 cell, a BHK-21 cell, and a HeLa cell. In another example, thepolypeptide, upon binding to the receptor, represses the Akt signalingtransduction pathway. In yet another example, the polypeptide, uponbinding to the receptor, activates procaspase-9, -7, or -3, whichfurther induces apoptosis.

As the polypeptide of this invention induces apoptosis, one thereforecan use it to induce death of a cell by contacting a cell with thepolypeptide. Thus, also within the scope of this invention are (i) apharmaceutical composition that contains the above-described polypeptideand a pharmaceutically acceptable carrier, and (ii) a method fortreating an apoptosis-related disorder in a subject, i.e., administeringto the subject an effective amount of the just-mentioned polypeptide.“An apoptosis-related disorder” refers to a condition characterized orcaused by an excessive level of cells. An excessive level refers to (1)a level higher than a normal level, and (2) a level higher than desiredin an individual, even though it is not greater than a normal level.Examples of the disorder include a cancer (e.g., breast cancer,colorectal cancer, leukemia, liver cancer, lung cancer, ovarian cancer,or prostate cancer), an infection by a virus (e.g., that by humanpapillomavirus, human immunodeficiency virus, or Hepatitis virus), anallergic disease, an inflammatory disease, an autoimmune disease, animmunodeficiency disease, a reperfusion injury, or a neurodegenerativedisorder.

This invention also features an isolated nucleic acid containing asequence encoding the above-described polypeptide. Examples of thenucleic acid include a sequence encoding SEQ ID NO: 1 (e.g., SEQ ID NO:2 listed above) and a sequence encoding SEQ ID NO: 7 (e.g., SEQ ID NO: 8listed below)

 532                                                        cgggacgtc 541 cgcgcacgaa acgcgccgtc gcttgaggaa cacttgtaca aacacgattt aagcaggttt 601 ccacaactga taaaactcgt gcaacttgaa actccgcctg gtctttccag gtctagaggg 661 gttacacttt gtactgtgct cgactccacg cccggtccac tggcgggtgt tagtagcagc 721 actgttgttt cgtagcggag catggtggcc gtgggaactc ctccttggtg acaagggccc 781 acggggccga aagccacgtc cagacggacc caccatgtgt gcaaccccag cacggcaact 841 tttactgcga acaccacctt aaggtgacac tggtactggt actcggtcac tggtgacagg 901 ctaaggatgc ccttcaggta ccccgaggta acacgggaca ctcgggatct gagaagggga 961 ttgggacttc tttaaaagtg cccagtttaa aaagcttcta cgcctgaata ggcgaccgga1021 ggccggcgcc tttccattac ccactactaa atccatgaat acgactgact gttttatcgc1081 tctgctatac gctctcagag agatcaaagc actgtttctg tcacgaacac aagggaagat1141 ggaattcaca ctttacaacg gtgaaaagaa ggtcttctac tccagaccca acaaccacga1201 caactgttgg ctgaacgcca tcctccaact gttcaggtac gttgacgagc ccttcctcga1261 atgggtctac gactcacctg agaacctcac tctcgaggcg atcaacaaac tggaagaaat1321 cacaggtctt gagctacacg agggcggacc gcccgccctt gtcgtctgga acatcaagca1381 cttgctctac accggaatcg gcaccgcttc gcgacccagc gaggtgtgca tggtggacgg1441 tacagacatg tgcttggctg acttccacgc cggtatattt ctgaagggac aggaccacgc1501 cgtcttcgcc tgcgtcacct ctgacgggtg gtacgcgatt gacgacgagg acttttaccc1561 gtggacacca aatccggccg acgttttggt ttttgttccg tacgatcaag aaccattcaa1621 cgcagaatgg aaagcaaagg ttcagaagcg gctcaggggc gccgggcaat ccagcccgac1681 gaccgggtca caaaaccaat ctggcaacac tggcagcatt attaacaatt actacatgca1741 gcagtaccag aactcaatgg acacccaact tggcgacaac gccattagtg gagggtccaa1801 cgagggctcc acggacacta cctctaccca caccaacaac acccagaaca acgactggtt1861 ttcgaaactg gccaacaccg cttttagcgg cctcttcggt gctcttcttg cagacaagaa1921 gacggaagaa accaccctcc tcgaagaccg catcctcacc acccgcaacg ggcacacgac1981 ctcgacaacc cagtctagcg tcggggtgac ttacgggtac gcaacggctg aagacttcgt2041 gagtgggcct aacacctctg gtcttgagac cagagttgtt caggccgaac ggttcttcaa2101 aacccacctg tttgactggg tcaccagtga cccgtttggg cggtgtcact tgttggagct2161 accgactgac cacaaaggcg tctacggtag cctgaccgac tcgtacgcat acatgaggaa2221 tggttgggac gttgaagtca ccgcagtggg taaccaattc aacggaggct gtttgctggt2281 ggcgatggta ccggagctct gttccatcag caagagagag ttgtaccagc ttacgctttt2341 cccccaccag ttcatcaacc cacggacgaa tatgacggca cacatcaccg tgccctacct2401 cggtgtcaac aggtacgacc agtacaaggt acacaaaccc tggaccctcg tggtcatggt2461 tgtggccccc ttgacggtta acaacgaggg cgctccgcaa atcaaggtgt atgccaacat2521 cgcccccacc aatgttcacg tcgcgggtga gctcccctct aaagagggga ttttccccgt2581 ggcatgcagc gatggttacg gtggcttggt gaccacggat ccgaagacgg cagaccccgt2641 ctacgggaaa gtgttcaacc caccccgcaa cctgttgcca gggcggttta caaacctcct2701 tgacgtggcc gaggcgtgcc ccacattcct acacttcgac ggtgacgttc cgtacgtgac2761 cacgaagacg gattcggata gggtgctagc ccagttcgat ttgtccctcg c

If the nucleic acid is operably linked to a regulatory sequence suitablefor expressing the polypeptide in host cells, it can express thepolypeptide after being introduced into the host cells. As thepolypeptides thus-expressed, upon binding to a cell-surface receptor,can kill cells, including host cells, the nucleic acid can also be usedfor inducing cell death or treating an apoptosis-related disorder in asubject.

An “isolated polypeptide” refers to a polypeptide that has beensubstantially separated from other proteins, lipids, and nucleic acidswith which it is naturally associated. The polypeptide can constitute atleast 50, 70, or 95% by dry weight of the purified preparation. An“isolated nucleic acid” refers to a nucleic acid the structure of whichis not identical to that of any naturally occurring nucleic acid or tothat of any fragment of a naturally occurring genomic nucleic acid. Theterm therefore covers, for example, (a) a DNA which has the sequence ofpart of a naturally occurring genomic DNA molecule but is not flanked byboth of the coding sequences that flank that part of the molecule in thegenome of the organism in which it naturally occurs; (b) a nucleic acidincorporated into a vector or into the genomic DNA of a prokaryote oreukaryote in a manner such that the resulting molecule is not identicalto any naturally occurring vector or genomic DNA; (c) a separatemolecule such as a cDNA, a genomic fragment, a fragment produced bypolymerase chain reaction (PCR), or a restriction fragment; and (d) arecombinant nucleotide sequence that is part of a hybrid gene, i.e., agene encoding a fusion protein. The nucleic acid of this invention canbe used to express a polypeptide of this invention. For this purpose,one can operatively link the nucleic acid to suitable regulatorysequences to generate an expression vector.

To produce a polypeptide of this invention, one can place a host cell ina culture under conditions permitting expression of a polypeptideencoded by a nucleic acid described above, and isolate the polypeptidefrom the culture. Alternatively, a nucleic acid of this invention can betranscribed and translated in vitro, for example, using T7 promoterregulatory sequences and T7 polymerase.

A vector refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked, and also capable ofautonomous replication or integration into a host DNA. Examples includea plasmid, cosmid, and viral vector. A vector of this invention includesa nucleic acid in a form suitable for expression of the nucleic acid ina host cell. Preferably, the vector includes one or more regulatorysequences operatively linked to the nucleic acid sequence to beexpressed. Examples of a regulatory sequence include promoters,enhancers, and other expression control elements (e.g., polyadenylationsignals). Regulatory sequences also include those that directconstitutive expression of a nucleotide sequence, as well astissue-specific regulatory and/or inducible sequences. The design ofsuch an expression vector is based on considerations including thechoice of the host cell to be transformed and the desired expressionlevel. An expression vector can be introduced into host cells to producea polypeptide of this invention. This invention also includes a hostcell that contains the above-described nucleic acid. The host cell canbe a bacterial cell, a yeast cell, an insect cell, a plant cell, and amammalian cell.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DETAILED DESCRIPTION

This invention is based, at least in part, on an unexpected discoverythat a water-soluble foot-and-mouth disease virus (FMDV) VP1 polypeptidepossesses apoptosis-inducing activity. The polypeptide and its variantsare useful for treating conditions associated with disorders caused byexcessive or unwanted cells.

Foot-and-mouth disease (FMD), a deadly epidemic, affects variouseconomically important domestic livestock including cattle, pigs, goats,and sheep (Woolhouse et al., 2001, Nature 411, 258-259). FMDV includesseven serotypes of viruses, all of which belong to the Aphthovirus genusof the family picomaviridae. The capsid of FMDV is made up of 60 copiesof each of four proteins, VP1, VP2, VP3, and VP4.

FMDV infects cells by attaching to cell-surface integrin through a long,conformationally flexible loop (G-H loop) of VP1 (Logan et al., 1993,Nature 362, 566-568). In some cases, it also uses heparin sulfate as analternative internalization receptor. The G-H loop contains a conservedarginine-glycine-aspartic acid (RGD) tripeptide motif that ischaracteristic of integrin ligands (See, e.g., Ruoslahti et al., 2003,Matrix Biol. 22, 459-465).

Integrin belongs to a family of cell surface α-β heterodimericglycoproteins. These proteins are responsible for a variety ofprocesses, including the induction of signal transduction pathways thatmodulate cell proliferation, morphology, migration and apoptosis (Hynes,1992, Cell 69, 11-25). Four species of integrin, i.e., α_(v)β₁, α_(v)β₃,α_(v)β₆, and α₅β₁, have been shown to mediate FMDV infection (Jackson,et al., 2000, J. Virol. 74, 4949-4956 and Jackson, et al., 2002, J.Virol. 76, 935-941). Although the VP1-integrin interaction mediates FMDVinfection, the study on VP1's biological effects is limited due to thepoor water solubility of VP1. Indeed, VP1 protein has been only usedtogether with denaturing agents such as urea, the presence of which hasmade it infeasible to evaluate the biological effects of VP1. Thus,there is a need for a water-soluble FMDV VP1 polypeptide.

This invention features a water-soluble FMDV VP1 polypeptide, as well asa nucleic acid encoding it. As mentioned above and described in theExample below, the water-soluble FMDV VP1 polypeptide, via binding tointegrin, induces apoptosis in certain cancer cells. It is known thatthe binding of a ligand to integrin activates the Akt signaltransduction pathway and protects cell from apoptosis (King, et al.,1997, Mol. Cell Biol. 17, 4406-4418 and Toker et al., 2000, Mol.Pharmacol. 57, 652-658). Thus, it is unexpected that the polypeptide ofthis invention binds to integrin and induces apoptosis. The polypeptideis useful for treating conditions associated with disorders caused byexcessive or unwanted cells.

A polypeptide of the invention can be obtained as a syntheticpolypeptide or a recombinant polypeptide. To prepare a recombinantpolypeptide, one can clone a nucleic acid encoding the polypeptide in anexpression vector, in which the nucleic acid is operably linked to aregulatory sequence suitable for expressing the polypeptide in a hostcell. One can then introduce the vector into a suitable host cell toexpress the polypeptide. Alternatively, the nucleic acid can be linkedto another nucleic acid encoding a fusion partner, e.g.,Glutathione-S-Transferase (GST), T7 tag, 6×-His epitope tag, M13 Gene 3protein, or an immunoglobulin heavy chain constant region. The resultantfusion nucleic acid expresses in suitable host cells a fusion protein.Suitable host cells are those that are resistant to this apoptoticpolypeptide and can be obtained using screening methods known in theart. The expressed recombinant polypeptides can be purified from thehost cell by methods such as ammonium sulfate precipitation andfractionation column chromatography. See Goeddel, (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.Water-soluble polypeptides are then prepared by the method described inU.S. application Ser. No. 10/449,531 and Wang et al., 2003, Vaccine 21,3721-3729t. An isolated fusion protein can be further treated, e.g., byenzymatic digestion, to remove the fusion partner and obtain therecombinant polypeptide of this invention.

The amino acid composition of a polypeptide of the invention may varywithout disrupting the ability of binding to integrin and inducingapoptosis. For example, such a variant can contain one or moreconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a polypeptide is preferably replaced with anotheramino acid residue from the same side chain family. Alternatively,mutations can be introduced randomly along all or part of a polypeptideof this invention, such as by saturation mutagenesis, and the resultantmutants can be screened for the ability of binding to integrin andinducing apoptosis to identify variants of this invention as descriedbelow in the example. Thus, as an example, the term “polypeptidecontaining SEQ ID NO: 1” covers polypeptides containing variants of SEQID NO: 1, including fusion proteins or proteins having one or moreconservative amino acid substitutions mutations, insertions, deletions,truncations, or combination thereof. Each variant retains substantiallythe activity of binding to integrin and inducing apoptosis.

Each of the above-described polypeptides can be tested for its apoptoticactivity on cells according to the method described in the Examplebelow. A polypeptide having apoptotic activity, as well as nucleic acidencoding it, can be used to induce cell death.

Thus, also within the scope of this invention is a method of inducingdeath of cells, e.g., by contacting cells with a polypeptide of theinvention in vitro, or by administering to a subject in need thereof aneffective amount of the polypeptide or nucleic acid, e.g., an expressionvector. Subjects to be treated can be identified as having or being atrisk for acquiring an apoptosis-related disorder.

The term “treating” refers to administration of a composition to asubject with the purpose to cure, alleviate, relieve, remedy, prevent,or ameliorate a disorder, the symptom of the disorder, the disease statesecondary to the disorder, or the predisposition toward the disorder. An“effective amount” is an amount of the composition that is capable ofproducing a medically desirable result in a treated subject. The methodcan be performed alone or in conjunction with other drugs or therapy.

Disorders to be treated include a disease caused by excessive abnormalcells (e.g., cancerous cells) or excessive normal cells (e.g., T-cells).Examples of a disease caused by excessive abnormal cells, i.e.,oncological disease, include retinoblastoma, Wilm's tumor, familialcolonic polyposis, hereditary non polyposis colon cancer,neurofibromatosis, familial chest cancer, xeroderma pigmentosum, blaincancer, oral cancer, esophageal cancer, stomach cancer, colon cancer,liver cancer, pancreatic cancer, lung cancer, thyroid cancer, mammarygland tumor, urinary tumor, virilia tumor, muliebria tumor, skin tumor,osteosarcoma, osteochondrosarcoma, leukemia, lymphoma, and solid tumor.Exemplary diseases caused by excessive T-cells include diabetesmellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, and psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, type Idiabetes, inflammatory bowel diseases, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, drug eruptions, leprosy reversal reactions, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis,uveitis posterior, interstitial lung fibrosis, graft-versus-hostdisease, cases of transplantation (including transplantation usingallogeneic or xenogeneic tissues) such as bone marrow transplantation,liver transplantation, or the transplantation of any organ or tissue,allergies such as atopic allergy, and AIDS.

In one in vivo approach, a therapeutic composition (e.g., a compositioncontaining a polypeptide of the invention or a nucleic acid encoding it)is administered to a subject. Generally, the polypeptide or nucleic acidis suspended in a pharmaceutically-acceptable carrier (e.g.,physiological saline) and administered orally or by intravenousinfusion, or injected or implanted subcutaneously, intramuscularly,intrathecally, intraperitoneally, intrarectally, intravaginally,intranasally, intragastrically, intratracheally, or intrapulmonarily.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thesubject's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100.0 mg/kg.Variations in the needed dosage are to be expected in view of thevariety of compositions available and the different efficiencies ofvarious routes of administration. For example, oral administration wouldbe expected to require higher dosages than administration by intravenousinjection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Encapsulation of the composition in a suitable delivery vehicle(e.g., polymeric microparticles or implantable devices) may increase theefficiency of delivery, particularly for oral delivery.

Also within the scope of this invention is a pharmaceutical compositionthat contains a pharmaceutically acceptable carrier and an effectiveamount of a polypeptide of the invention or a nucleic acid encoding it.The pharmaceutical composition can be used to treat diseases describedabove. The pharmaceutically acceptable carrier includes a solvent, adispersion medium, a coating, an antibacterial and antifungal agent, andan isotonic and absorption delaying agent.

The pharmaceutical composition of the invention can be formulated intodosage forms for different administration routes utilizing conventionalmethods. For example, it can be formulated in a capsule, a gel seal, ora tablet for oral administration. Capsules can contain any standardpharmaceutically acceptable materials such as gelatin or cellulose.Tablets can be formulated in accordance with conventional procedures bycompressing mixtures of the composition with a solid carrier and alubricant. Examples of solid carriers include starch and sugarbentonite. The composition can also be administered in a form of a hardshell tablet or a capsule containing a binder, e.g., lactose ormannitol, a conventional filler, and a tableting agent. Thepharmaceutical composition can be administered via the parenteral route.Examples of parenteral dosage forms include aqueous solutions, isotonicsaline or 5% glucose of the active agent, or other well-knownpharmaceutically acceptable excipient. Cyclodextrins, or othersolubilizing agents well known to those familiar with the art, can beutilized as pharmaceutical excipients for delivery of the therapeuticagent.

The efficacy of a composition of this invention can be evaluated both invitro and in vivo. See, e.g., the examples below. Briefly, thecomposition can be tested for its ability to induce death of cells invitro. For in vivo studies, the composition can be injected into ananimal (e.g., a mouse model) and its therapeutic effects are thenaccessed. Based on the results, an appropriate dosage range andadministration route can be determined.

The specific example below is to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications recited herein arehereby incorporated by reference in their entirety.

EXAMPLE VP1 Induced Apoptosis

To examine whether VP1 induces apoptosis, aqueous soluble recombinantVP1 (rVP1) was expressed in E. coli and purified according to the methoddescribed in U.S. application Ser. No. 10/449,531 and Wang et al., 2003,Vaccine 21, 3721-3729. Since aqueous soluble rVP1 might form monomers ordimers depending upon redox conditions during experiments, a mutant rVP1monomer was generated by changing the position 201 cysteine to serinevia site-directed mutagenesis following the protocol described in Du etal., 1995, Biotechniques 18, 376-378. The mutated VP1 gene was ligateinto the expression vector pET24a(+). The mutant and wild type proteinswere expressed in E. coli and purified in the same manner described inthe aforementioned two references.

Baby hamster kidney fibroblast cells (BHK-21) were used to examine theapoptosis-inducing activity of wild type and mutant rVP1. BHK-21 cellsare known to be permissive for FMDV to bind to through the RGD motif andreplicate (Baxt et al., 1984, J. Virol. 51, 298-305). More specifically,BHK-21 cells were maintained at 37° C. in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mML-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cellswere plated at 2×10⁵ cells per well in a twelve-well plate (400μl/well). One day later, the cells were washed twice withphosphate-buffered saline (PBS) and incubated with a FCS-free DMEMcontaining 1 μM rVP1. The cells were then incubated at 37° C. overnightbefore being examined for DNA fragmentation, a hallmark of apoptosis(Slee, et al., 1999. J. Cell Biol. 144, 281-292).

Briefly, the BHK-21 cells treated with rVP1 were washed by ice-cold PBSand transferred to centrifuge tubes. The cells in each tube wererecovered by centrifugation at 1,500×g for 10 minutes at 4° C. andre-suspended in 100 μl of a solution containing 10 mM Tris-Cl and 1 mMEDTA, pH 8.0. The suspension was mixed with 1 ml of an extraction buffercontaining 0.1 M EDTA, 0.5% (w/v) SDS, 20 μg/ml pancreatic RNase and 10mM Tris-Cl, pH 8.0 and incubated at 37° C. for 1 hour. Proteinase K(Sigma) was then added to a final concentration of 100 μg/ml and thesuspension of lysed cells was incubated at 50° C. for 3 hours withperiodically swirling. An equal volume of phenol was then added to thesuspension. The mixture was kept at room temp for 10 minutes and thencentrifuged at 5,000×g for 15 minutes to separate into two phases. Afterthree times extraction with phenol, the aqueous phases were pooled,transferred to a centrifuge tube, and mixed with 0.2 volume of 10 Mammonium acetate as well as 2 volumes of ethanol. The genomic DNA wascollected by centrifugation at 5,000×g for 5 minutes, washed with 70%ethanol, separated by electrophoresis on a 0.8% agarose gel, andexamined for DNA fragmentation.

It was found that genomic DNA prepared from BHK-21 cells treated withwild type or mutant rVP1 was fragmented to about the same degree. Thisresult indicates that both the with wild and mutant rVP1 proteins caninduce apoptosis in BHK-21 cells. The mutated monomeric rVP1 was used inall following experiments.

BHK-21 cells were further used to evaluate whether the above-mentionedDNA fragmentation was accompanied by cell death and DNA condensation.Briefly, BHK-21 cells (5×10⁵ cells ml⁻¹) were transfected with a plasmidencoding green fluorescent protein gene (pEGFP-C3, 0.5 μg/well, BDBiosciences, Palo Alto, Calif.) in Effectene (Qiagen, Valencia, Calif.)for 24-48 hours. The cells were then incubated with a medium containing0 (control) or 1.0 μM rVP1 for 2 or 24 hours. The living cells, whichemitted green fluorescent upon excitation, were identified and countedunder a fluorescence microscope before and after the rVP1 incubation. Itwas found that the number of living cells was reduced after 2 hours ofincubation and further reduced after 24 hours of incubation.

To visualize DNA condensations in nuclei, BHL-21 cells were treated with0 (control), 0.05, 0.1, 0.5, 1.0, or 3.0 μM rVP1 for 16 hours. The cellswere then stained with DAPI (0.5 μg/ml in PBS, Sigma, St. Louis, Mo.)according to the manufacturer's instructions and examined by lightmicroscopy. The level of apoptosis was determined as the ratio of thecells with morphologically changed nuclei to the total number of cellspresent, i.e., apoptotic index. It was found that apoptotic indexes ofthe cells treated with 0.1, 0.5, 1.0, and 3.0 μM rVP1 were about 10%,90%, 95%, and 99%, respectively. In contrast, the apoptotic index of thecontrol cells was less than 1%. These results indicate that rVP1 indeedcauses apoptosis.

To compare the apoptotic effect of rVP1 with those of other RGDcontaining molecules, BHK-21 cells were treated with rVP1, cyclic RGD,or P29 peptide (peptide containing sequence of amino acid residues of139-164 with RGD motif at position 145-147) in the manner descriedabove. It was found that the treatment by 1 μM rVP1 induced apoptosis inalmost all BHK-21 cells, while cyclic RGD at the same concentration hadlittle effect. The 50% effective concentration of rVP1 in causingapoptosis was around 0.3 μM which was about 100 fold and 10,000 foldmore potent than that of the P29 peptide (30 μM) and cyclic RGD (3 mM),respectively.

VP1 Induced Apoptosis via Binding to Integrin

The above-described experiments were repeated except that, beforeincubating with rVP1, the cells were incubated with rabbit anti-FMDVneutralizing antibodies (RN) or mouse anti-integrin VLA-5 (α₅β₁)monoclonal antibody (Chemicon, Temecula, Calif.). These antibodies areknown to block the binding of integrin to its ligand. It was found thatthe apoptotic effect of rVP1 was blocked specifically by these twoantibodies. It is known that fibronectin is a natural ligand forintegrin α₅β₁. The effects of fibronectin on rVP1-induced apoptosis inBHK-21 cells were investigated in the manner described above. The resultshowed that fibronectin at 0.8 μM abolished the apoptotic effect of 1 μMrVP1. Both of the above results indicate that rVP1 induces apoptosis viabinding to integrin.

To further demonstrate that the apoptotic effect of rVP1 requires theinteraction between rVP1 and integrin, rVP1 was expressed in BHK-21cells intracellularly. More specifically, BHK-21 cells were transfectedwith 0.3 μg plasmid pIBSY1 or pIBSY1-VP1, an expression vector encodingVP1 (Shieh, et al., 2001, Vaccine 19, 4002-4010). The transfected cellswere subjected to apoptosis assays in the same manner described above.It was found that, although rVP1 was significantly expressed in thecells transfected with pIBSY1-VP1, little apoptosis was observed evenafter 3-days of culture. On the other hand, addition of rVP1 (0.5 μM) tothe culture medium resulted in the death of the transfected cells. Theseresults suggest that induction of apoptosis by rVP1 is due to itsbinding to extracellular domains of integrin.

It is known that integrin signaling activates the PI3-K/Akt signaltransduction pathway, an anti-apoptotic mechanism utilized by many typesof cells (King et al., 1997, Mol. Cell Biol. 17, 4406-4418 and Toker2000, Mol Pharmacol 57, 652-658. Akt, a serine/threonine kinase, canbind to phosphorylated lipids at the membrane in response to theactivation of phosphatidylinositol 3-kinase (PI3-kinase) by growthfactors and mediates cell survival. Phosphorylated Akt activatesanti-apoptotic or inhibits pro-apoptotic processes in a cell byphosphorylating Bad, Forkhead transcription factors, glycogen synthasekinase 3β (GSK-3β), and caspase-9 (See, e.g., Benetti et al., 2003, J.Viro. 1 77, 6567-6573). Deactivation of Akt, on the other hand,activates pro-apoptotic responses (Luo, et al., 2003, Proc. Natl. Acad.Sci. USA 100, 11712-11717). Both GSK-3,3 and caspase-9 are pro-apoptoticfactors. Cleavage of caspase-9, a cysteine aspartic acid protease,results in induction of a caspase cascade, including the processing ofprocaspase-3 and -7, which eventually leads to apoptosis. Activation ofPI3-kinase through G protein-coupled receptors such assphingosine-1-phosphate (S1P) receptors or PI3-kinase inhibitors (e.g.,LY294002) modulates the activity of Akt. Thus, PI3-K/Akt signaltransduction pathway, GSK-3β, and caspases were further analyzed.

VP1 Treatment Deactivated Akt

In this experiment, the PI3-K/Akt signal transduction pathway wasexamined in BHK-21 cells treated with rVP1. As binding of PDGF to cellsactivates the pathway and causes cell proliferation (Slee, et al., 1999,J. Cell Biol. 144, 281-29228, and Schneller et al., 1997, Embo. J. 16,5600-5607), BHK-21 cells were incubated with PDGF (PeproTech EC LTD,London, United Kingdom) and rVP1, respectively.

More specifically, BHK-21 cells were incubated with rVP1 in the mannerdescribed above in presence or absence of PDGF (0.1 μg). At minutes 5,30, and 60, the cells were lysed in 0.2 ml of a boiling protein loadingbuffer (Invitrogen). Twenty microliter of each boiled sample wasanalyzed for Akt and GSK-3β phosphorylation by Western blotting usingprimary antibodies against Akt, phospho-Akt (Ser473), and phospho-GSK-3β(Ser9), as well as anti-actin, anti-integrin VLA-5 (α₅β₁) monoclonalantibody, and anti-mouse immunoglobulin G horseradish peroxidase-coupledsecondary antibodies. The antibodies against Akt, phospho-Akt (Ser473),and phospho-GSK-3β (Ser9) were obtained from Cell Signaling Technology,Inc (Beverly, Mass.). Anti-actin, anti-integrin VLA-5 (α₅β₁) antibody,and anti-mouse immunoglobulin G horseradish peroxidase-coupled secondaryantibodies were obtained from Chemicon (Temecula, Calif.).

As expected, PDGF activated Akt phosphorylation, and this effect wasinhibited by 10 μM PI-3K inhibitor LY294002 (Cell Signaling Technology,Inc, Beverly, Mass.). It was found that rVP1 incubation inhibited Aktphosphorylation in a dose dependent manner after BHK-21 cells wereincubated with rVP1 for 30 minutes. This inhibitory effect was reversedby pre-treatment of cells with anti-integrin α₅β₁ antibodies for 30 min,indicating that integrin was required for the inhibition.

Phosphorylation of GSK-3β was also examined according to the methoddescribed in Cross et al., 1995, Nature 378, 785-789. It was found thatrVP1 inhibited the phosphorylation of GSK-3β in a dose dependent manner.

To verify that the level of phosphorylated GSK-3β correlated positivelywith BHK-21 cell survival, BHK-21 cells were treated with GSK-3βinhibitor (GSK-3βI, Calbiochem) thereby inhibiting the formation ofGSK-3β according to the method described in Coghlan et al., 2000, Chem.Biol. 7, 793-803. The results showed that treatment of BHK-21 cells withGSK-3βI attenuated the apoptotic effect of rVP1 in a dose-dependentmanner, indicating that rVP1 induces apoptosis via GSK-3β.

VP1 Activated Procaspase Cleavage

Caspases, a family of cysteine aspartic acid proteases, are centralregulators of apoptosis (Slee et al., 1999, J. Cell Biol. 144, 281-292).Fragmentation of DNA causes cleavage of procaspase-9, which in turnresults in processes of other procaspases, such as procaspase-3 andprocaspase-7. The initiation of the caspase cascade eventually leads toapoptosis. Thus, the effects of rVP1 treatment on the activation ofprocaspase-9 and downstream procaspases-7 and -3 were evaluated.

Pprocaspases-3, -7, and -9 were obtained from Cell Signaling Technology,Inc (Beverly, Mass.). BHK-21 cells were treated with increasing amountsof rVP1 (0, 0.1, 0.2, and 0.5 μM) in the same manner described above.After incubation for 16 hours, the cells were lysed. The resultantlysates were analyzed by Western blot using antibodies againstprocaspases-3, -7, and -9.

It was found that the rVP1 treatment caused the cleavage ofprocaspases-9, -7 and -3. Cleavage of both procaspases-9 and -7 wasdetected in BHK-21 cells treated with 0.1 μM of rVP1 and became morepronounced at 0.5 μM rVP1. Cleavage of procaspase-3 was not detectableuntil higher concentrations (≧0.5 μM) of rVP1 were used. These resultsindicate that rVP1 induces cleavage of procaspases.

VP1 Induced Apoptosis in Cancer Cells

The aforementioned Akt signal transduction pathway is a major target fortreatment of all four major human cancers, i.e., breast, prostate, lung,and colorectal cancers (See, e.g., Roy et al., 2002, Carcinogenesis 23,201-205). In the following experiments, the effects of rVP1 on Aktsignal transduction pathway in cancer cells were examined.

First, the effects of rVP1 on the Akt signal transduction pathway wasevaluated in breast carcinoma MCF-7 cells. More specifically, MCF-7cells were incubated with rVP1 in presence or absence of GSK-3βinhibitor I (Calbiochem) in the same manner described above. It wasfound that (1) rVP1 caused apoptosis in MCF-7 cells in a dose-dependentmanner (2) this rVP1-induced apoptosis was reversed by GSK-3β inhibitorI in a dose-dependent manner. As MCF-7 cells lack a functional caspase-3gene, these results suggest that the apoptotic effect of rVP1 isindependent of caspase-3 and differ from that of Buckley et al., 1999,Nature 397, 534-539, which showed that seven RGD-containing polypeptidewere unable to induce apoptosis in MCF-7 cells. These results alsosuggest that the VP1 polypeptide and its functional equivalents aredifferent form the Buckley polypeptides and can be used to treat breastcancer. Indeed, none of the Buckley polypeptides contains RGDL.

Second, prostate cancer cell lines PC-3 and 22Rv1 were used to examinethe apopototic effects of rVP1. As mentioned above, activation ofPI3-kinase through S1P receptors modulates Akt activity. In fact,binding of S1P to S1P receptors subtype 3 (S1P₃) mediates Akt activationand crosstalk with PDGF receptor. It is known that 22Rv1 cells expressS1P₃ and are responsive to androgen stimulation. In contrast, PC-3 cellsdo not express S1P3 and are non-responsive to androgen (Baudhuin, etal., 2004, Faseb. J. 18, 341-343). The two types of cells weremaintained at 37° C. in DMEM-10% FCS containing 2 mM L-glutamine, 100U/ml penicillin, and 100 mg/ml streptomycin. The cells were plated at2×10⁵ cells per well in a twelve-well plate (400 μl/well). One daylater, the cells were incubated with media containing 0, 0.2, 0.4, 0.8,and 1.2 μM of rVP1, respectively, and subjected to apoptosis assays inthe manner described the experiments. The results showed that rVP1caused apoptosis in both cell types and exerted more apoptotic effect onPC-3 cells. These results suggest that the apoptotic effect of rVP1 isnot via deactivation of S1P₃. Further, they suggest that rVP1 can beused to treat androgen-independent prostate cancers, such as PC-3, whichare usually much more difficult to cure than androgen-dependent prostatecancers.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of theinvention.

1. A method for treating an apoptosis-related disorder in a subject, themethod comprising administering to a subject in need thereof aneffective amount of a water-soluble VP1 polypeptide of foot-and-mouthdisease virus or a nucleic acid encoding the polypeptide, wherein theVP1 polypeptide contains RGD, binds to integrin, and induces cell death.2. The method of claim 1, wherein the VP1 polypeptide contains an aminoacid sequence of SEQ ID NO:1.
 3. The method of claim 1, wherein the VP1polypeptide contains an amino acid sequence of SEQ ID NO:3.
 4. Themethod of claim 1, wherein the apoptosis-related disorder is selectedfrom the group consisting of cancer, viral infection, allergic disease,inflammatory disease, autoimmune disease, immunodeficiency disease,reperfusion injury, and neurodegenerative disorder.
 5. The method ofclaim 4, wherein the VP1 polypeptide contains an amino acid sequence ofSEQ ID NO:1.
 6. The method of claim 4, wherein the VP1 polypeptidecontains an amino acid sequence of SEQ ID NO:3.
 7. The method of claim4, wherein the apoptosis-related disorder is cancer.
 8. The method ofclaim 7, wherein the cancer is selected from the group consisting ofbreast cancer, leukemia, liver cancer, lung cancer, ovarian cancer, andprostate cancer.
 9. The method of claim 7, wherein the VP1 polypeptidecontains an amino acid sequence of SEQ ID NO:1.
 10. The method of claim7, wherein the VP1 polypeptide contains an amino acid sequence of SEQ IDNO:3.
 11. The method of claim 7, wherein the cancer is liver cancer. 12.The method of claim 11, wherein the VP1 polypeptide contains an aminoacid sequence of SEQ ID NO:1.
 13. The method of claim 11, wherein theVP1 polypeptide contains an amino acid sequence of SEQ ID NO:3.
 14. Themethod of claim 7, wherein the cancer is ovarian cancer.
 15. The methodof claim 14, wherein the VP1 polypeptide contains an amino acid sequenceof SEQ ID NO:1.
 16. The method of claim 14, wherein the VP1 polypeptidecontains an amino acid sequence of SEQ ID NO:3.